Abstract Cells must maintain a balance between generating, folding, transporting, and degrading proteins in order to maintain proper protein homeostasis, or proteostasis. A central player in the maintenance of mammalian proteostasis is p97, a AAA+ ATPase (ATPase associated with diverse cellular activities) that leverages the power of ATP hydrolysis to pull ubiquitinated substrates from a variety of organelles and unfold them before proteasomal degradation. Mutations in p97 can lead to diseases associated with dysregulation of proteostasis; thus, while it is known that p97 is critical to cellular health, much about its mechanism remains unknown. In general, p97 must bind, translocate, and release the unfolded substrate. Each of these steps is dependent on p97’s interactions with multiple binding partners, yet how these interactions are coordinated has not been fully characterized. Studies have shown that the p97 binding partner, Otu1, trims ubiquitin moieties from p97 substrates to allow their efficient unfolding and release. Yet how this deubiquitination occurs remains an open question. Solving the p97-Otu1 structure will elucidate how polyubiquitinated substrates are deubiquitinated and will construct a more complete understanding of how p97 processes its substrates. Recent work has explored unfolding initiation in the context of the heterodimeric Ufd1/Npl4 (UN) binding partner. However, it is not known if this initiation mechanism extends to other polyubiquitin substrate recruiting binding partners or if they utilize a unique mechanism. One of the most important p97 binding partners is p47, which is involved in Golgi membrane remodeling. Historically, p47 was reported to only interact with specific non-ubiquitinated or monoubiquitinated proteins; however, recent evidence has also demonstrated that p47 also interacts with polyubiquitinated substrates. Exploring the structural and biochemical basis of this action would broaden understanding of how p97 complexes unfold polyubiquitinated substrates. To elucidate how p97 is regulated by binding partners and how those binding partners interact with polyubiquinated substrate, I will determine high-resolution structures of the p97-Otu1 complex and the p97-p47 complex in complex with polyubiquitinated substrates via cryo-EM. Under the support of this grant, I plan to gain expertise in biochemistry and structural biology, improve my mentoring skills, refine my science communication, and become a leader with sensitivity for those that also come from underrepresented backgrounds. Under the mentorship of Dr. Peter Shen, and with the cutting-edge resources provided by the University of Utah, I am confident that the training plan will maximize my development towards my future career as an independent scientist.

Project Narrative The enzyme p97 is an abundant and conserved molecular machine that unfolds proteins prior to their degradation. p97 is regulated by many binding partners through unknown mechanisms. The proposed research will investigate how binding partners regulate p97, which will inform how p97 complexes function in maintaining cellular health and how mutations cause imbalance in protein homeostasis.